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Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions.

Zhang Z, Hui E, Chapman ER, Jackson MB - Mol. Biol. Cell (2010)

Bottom Line: Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly.All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood.By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA.

ABSTRACT
Synaptotagmin (syt) serves as a Ca(2+) sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca(2+) triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

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Syt I mutations and isoforms affect secretion rate. (A) Sample amperometry traces for wild-type syt I and two syt I mutants. The thick horizontal line below indicates the time of depolarization by a puff of high KCl. (B) Cumulative spike counts were plotted versus time to illustrate the time course of fusion triggered by depolarization (starting at t = 0). (C) Secretion rates for the different mutants were computed as number of spikes in the first 20 s. Amperometry traces for different syt isoforms (D), cumulative spike plots (E), and secretion rates (F). Data were from 42 to 125 cells. *, p < 0.05; **, p < 0.01. Error bars represent SEM.
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Figure 2: Syt I mutations and isoforms affect secretion rate. (A) Sample amperometry traces for wild-type syt I and two syt I mutants. The thick horizontal line below indicates the time of depolarization by a puff of high KCl. (B) Cumulative spike counts were plotted versus time to illustrate the time course of fusion triggered by depolarization (starting at t = 0). (C) Secretion rates for the different mutants were computed as number of spikes in the first 20 s. Amperometry traces for different syt isoforms (D), cumulative spike plots (E), and secretion rates (F). Data were from 42 to 125 cells. *, p < 0.05; **, p < 0.01. Error bars represent SEM.

Mentions: Syt I(R399A), with its mutation on the surface of the C2B domain opposite of the lipid-penetrating loops (Figure 1), does not stimulate SNARE-mediated Ca2+-triggered liposome fusion as effectively as wild-type syt I, but its binding to t-SNAREs is comparable to wild type (Gaffaney et al., 2008). We found that overexpression of syt I(R399A) in PC12 cells reduced the secretion rate by 41% (Figure 2, A–C; 1.06 ± 0.10 spikes/s versus the rate of 1.81 ± 0.16 spikes/s in cells overexpressing wild-type syt I; p < 0.01) and decreased the PSF lifetime by 18% (Figure 3, B and C; 1.10 ± 0.06 msec versus 1.34 ± 0.06 with wild-type syt I; p < 0.01). Syt I(T328A) provides a contrast to syt I(R399A). Its mutation resides on the side of the C2B domain close to the lipid-penetrating loops (Figure 1). Syt I(T328A) binds t-SNAREs more strongly than wild-type syt I but fails to promote liposome fusion as efficiently as wild-type syt I (Gaffaney et al., 2008). Overexpression of syt I(T328A) in PC12 cells reduced the secretion rate by 33% (Figure 2, A–C; 1.22 ± 0.15 spikes/s, p < 0.05) but left the fusion pore lifetime unaltered (Figure 3, B and C, 1.25 ± 0.05 msec, p > 0.05).


Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions.

Zhang Z, Hui E, Chapman ER, Jackson MB - Mol. Biol. Cell (2010)

Syt I mutations and isoforms affect secretion rate. (A) Sample amperometry traces for wild-type syt I and two syt I mutants. The thick horizontal line below indicates the time of depolarization by a puff of high KCl. (B) Cumulative spike counts were plotted versus time to illustrate the time course of fusion triggered by depolarization (starting at t = 0). (C) Secretion rates for the different mutants were computed as number of spikes in the first 20 s. Amperometry traces for different syt isoforms (D), cumulative spike plots (E), and secretion rates (F). Data were from 42 to 125 cells. *, p < 0.05; **, p < 0.01. Error bars represent SEM.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921110&req=5

Figure 2: Syt I mutations and isoforms affect secretion rate. (A) Sample amperometry traces for wild-type syt I and two syt I mutants. The thick horizontal line below indicates the time of depolarization by a puff of high KCl. (B) Cumulative spike counts were plotted versus time to illustrate the time course of fusion triggered by depolarization (starting at t = 0). (C) Secretion rates for the different mutants were computed as number of spikes in the first 20 s. Amperometry traces for different syt isoforms (D), cumulative spike plots (E), and secretion rates (F). Data were from 42 to 125 cells. *, p < 0.05; **, p < 0.01. Error bars represent SEM.
Mentions: Syt I(R399A), with its mutation on the surface of the C2B domain opposite of the lipid-penetrating loops (Figure 1), does not stimulate SNARE-mediated Ca2+-triggered liposome fusion as effectively as wild-type syt I, but its binding to t-SNAREs is comparable to wild type (Gaffaney et al., 2008). We found that overexpression of syt I(R399A) in PC12 cells reduced the secretion rate by 41% (Figure 2, A–C; 1.06 ± 0.10 spikes/s versus the rate of 1.81 ± 0.16 spikes/s in cells overexpressing wild-type syt I; p < 0.01) and decreased the PSF lifetime by 18% (Figure 3, B and C; 1.10 ± 0.06 msec versus 1.34 ± 0.06 with wild-type syt I; p < 0.01). Syt I(T328A) provides a contrast to syt I(R399A). Its mutation resides on the side of the C2B domain close to the lipid-penetrating loops (Figure 1). Syt I(T328A) binds t-SNAREs more strongly than wild-type syt I but fails to promote liposome fusion as efficiently as wild-type syt I (Gaffaney et al., 2008). Overexpression of syt I(T328A) in PC12 cells reduced the secretion rate by 33% (Figure 2, A–C; 1.22 ± 0.15 spikes/s, p < 0.05) but left the fusion pore lifetime unaltered (Figure 3, B and C, 1.25 ± 0.05 msec, p > 0.05).

Bottom Line: Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly.All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood.By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA.

ABSTRACT
Synaptotagmin (syt) serves as a Ca(2+) sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca(2+) triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

Show MeSH
Related in: MedlinePlus